Splicing of primary transcript is an essential and regulated step in the generation of functional mRNAs. Many human genes express two or more mRNAs via alternative splicing of their primary transcript, and disruption of normal splicing patterns is often associated with disease. Usage of alternative splice sites is achieved through the action of regulatory proteins, which promote or block the recruitment of constitutive splicing factors, or influence their function. Understanding the mechanisms that ensure efficient and accurate recognition of splice sites demands a detailed analysis of the core splicing machinery. Our studies focus on the second transesterification step and product release. These steps depend on the sequential action of the DEAH-box NTPases Prp16, Prp22 and Prp43. Biochemical dissection of the molecular interactions required for step 2 is essential for understanding how 3'splice site choice can be altered, either by regulatory proteins during alternative splicing or by disease-causing mutations. Defects in spliceosome disassembly steps are expected to influence subsequent rounds of spliceosome assembly and catalysis, insofar as some limiting splicing factors will be sequestered in "product complexes" and fail to recycle. Upon release from the spliceosome, the 2',5'phosphodiester bond in the lariat-intron RNA is "debranched" by a specialized enzyme, Dbr1. We are investigating the mechanism by which Dbr1 specifically recognizes and cleaves branched RNA. Dbr1 plays an important role for the turnover of introns, which comprise a large portion of the transcriptosome and serve as reservoirs for non-coding small RNAs. Project Narrative: This project addresses the mechanism of mRNA splicing, a fundamental step in gene expression. Defects and in this process can alter the structure and function of a gene product thus lead to disease. Understanding the basic mechanism of mRNA splicing is critical to understand how defects can lead to disease.